Isolation and cell-free translation of a human immunoglobulin light chain messenger RNA

Yaffe, Lyn; Pestka, Sidney

doi:10.1016/0003-9861(78)90303-x

Cited by 4 publications

(2 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…As can be seen in Table 1, the hybrid aD5-DH11-BC11 secreted 4-6 times more human u chain than did the human B cell lines GM607 or SED. We recovered [6][7] These observations and the lack of functional endoplasmic reticulum and Golgi apparatus in reticulocyte lysates, combined with the known 10-12% carbohydrate component of human ,u chains secreted in vivo (2) all are consistent with the absence of glycosylation of the in vitro synthesized ,u polypeptides and their faster mobility on reducing gels. Furthermore, mouse H chains contain an additional NH2-terminal signal peptide and still show similar mobility relationships between in vitro and in vivo synthesized polypeptides (29).…”

Section: Resultsmentioning

confidence: 62%

See 1 more Smart Citation

Cloning and partial nucleotide sequence of human immunoglobulin mu chain cDNA from B cells and mouse-human hybridomas.

Dolby¹,

Devuono²,

Croce³

1980

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Purified mRNAs coding for # and K human immunoglobulin polypeptides were translated in vitro and their products were characterized. The p-specific mRNAs, derived from both human lymphoblastoid cells (GM607) and from a mouse-human somatic cell hybrid secreting human p chains (aD5-DH11-BC11), were copied into cDNAs and inserted into the plasmid pBR322. Several recombinant cDNAs that were obtained were identified by a combination of colony hybridization with labeled probes, in vitro translation of plasmidselected p mRNAs, and DNA nucleotide sequence determination. One recombinant DNA, for which the sequence has been partially determined, contains the codons for part of the C3 constant region domain through the carboxy-terminal piece (155 amino acids total) as well as the entire 3' noncoding sequence up to the poly(A) site of the human p mRNA. The sequence A-A-U-A-A occurs 12 nucleotides prior to the poly(A) addition site in the human p mRNA. Considerable sequence homology is observed in the mouse and human p mRNA 3' coding and noncoding sequences.

show abstract

Section: Resultsmentioning

confidence: 62%

“…Virtually nothing of this nature has been reported for the human immunoglobulin gene system other than some early attempts at purification and in vitro translation of the ,u and X chain mRNAs (6,7).…”

mentioning

confidence: 99%

Cloning and partial nucleotide sequence of human immunoglobulin mu chain cDNA from B cells and mouse-human hybridomas.

Dolby¹,

Devuono²,

Croce³

1980

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

Protein synthesis in cell-free reticulocyte lysates on multi-hour incubation

Findeis

Whitesides

1987

Appl Biochem Biotechnol

View full text Add to dashboard Cite

Cell-free protein synthesis in rabbit reticulocyte lysate translation mixtures has been studied during multi-hour incubations. In an impaired lysate obtained from cells stored at 0'C before lysis, and showing a low initial rate of synthesis, translation could be stimulated by a factor of 4by including RNase inhibitor and additional ATP and GTP. In translation mixtures prepared from normal lysates, protein svnthesis could be improved by -50oh by the addition of excess GTP. The observed increases in protein synthesis were obtained by improved maintenance of the initial rate of synthesis.Index Entries: Cell-free protein synthesis; cell-free translation; reticulocyte lysate, and behavior on multi-hour incubation. INTRODqCTIONThe in vitro, cell-free synthesis of proteins is a much more difficult enterprise than in vivo synthesis using recombinant DNA technology, but it is a technique of real potential utility for the synthesis of proteins containing labeled or unnatural aminoacids, and for synthesis of proteins containing certain types of unnatural amino acid sequences. Chemical methods work well for the synthesis of relatively small peptides ( < 40 amino acid residues in length) but lose efficiency as synthesis of larger products is pursued (1,2). For larger proteins (MW>20,000), chemical synthesis may never be able to rival the combination of rDNA methods coupled with fermentation.

show abstract